Kaposi's sarcoma-associated herpesvirus (KSHV), also called Human herpesvirus type 8, is associated with Kaposi's sarcoma (KS) and two lymphoproliferative diseases: primary effusion lymphoma (PEL) and a subset of Multicentric Castleman's disease (MCD) (1-3). LANA is a multifunctional protein, which regulates host and viral gene expression and is the only viral protein required for latent DNA replication and episome segregation during latency. Many mechanistic details about LANA's role, latent DNA replication, episome tethering and transcription have been revealed. However, the complex molecular events leading to the establishment of latency, which allow viral episomes to be stably maintained in an epigenetic configuration permissive to reactivation, are still poorly understood. We hypothesize that early events after infection are crucial to establish latency during which viral episomes are replicated and segregated in a LANA-dependent fashion. We found that LANA specifically interacts with SSRP1 a chromatin remodeling factor that is important for latent DNA replication. LANA interaction with SSRP and other known epigenetic modifiers suggest that LANA functions in shaping both the viral and host cellular epigenomes. In aim 1, we propose to further study SSRP1/LANA interaction and its possible role in chromatin remodeling. In aim 2, we propose to first generate a detailed genome-wide map of the KSHV epigenome in latently infected cells and then to follow the occurrence of these epigenetic modifications after de novo infection of endothelial cells. SA1: Determine the molecular mechanisms by which LANA supports latent DNA replication through interactions with chromatin remodeling factors. Based on our recent finding that LANA specifically interacts with SSRP1, a subunit of FACT, we will map LANA and SSRP1 interaction domains and determine whether this complex also contributes to LANA-dependent transcriptional regulation. In addition, we will utilize reconstituted histone chromatin preparations to determine whether LANA/SSRP1 complexes have nucleosome disassembly activity. We also identified several additional LANA/origin interacting cellular proteins and will determine whether they play a role in LANA-dependent DNA replication and latency. SA2: Study LANA's contribution to the epigenetic state of the viral genome and transcriptional regulation of both host and viral genes during the establishment of latency. Using chromatin immunoprecipitations (ChIPs) in combination with High Throughput Sequencing (HTS), we propose to study histone modifications, pol II status, and LANA occupancy in PEL cells and long-term infected SLK and TIVE cells with high resolution on a genome-wide scale. We also propose to perform a genome-wide Methyltransferase Accessibility Protocol for Individual Templates (MAP-IT) analysis, a novel technique which probes methylation status, chromatin accessibility, and nucleosome positioning on a single molecule basis when combined with HTS sequencing. After first characterizing the KSHV epigenome during latency in cells of lymphoid and endothelial origin, we propose to study how the latent KSHV epigenome is established after de novo infection of endothelial TIVE and SLK cells. In summary, this highly innovative approach is aimed at increasing our understanding of what constitutes a latent genome and how LANA contributes to the complex events leading to the establishment of latency, a central question to both the biology and pathogenesis of KSHV.